JP6057418B2 - Method for obtaining a cell culture comprising hepatocytes from a group of cells containing hepatocytes differentiated from induced pluripotent stem cells - Google Patents

Description

  The present invention relates to a method of obtaining a cell culture consisting essentially of hepatocytes from a group of cells containing hepatocytes induced to differentiate from induced pluripotent stem cells, and cells substantially consisting of hepatocytes obtained by the method Relates to the culture. More specifically, the present invention relates to a method for obtaining a cell culture consisting essentially of hepatocytes from a cell group comprising hepatocytes induced to differentiate from induced pluripotent stem cells and undifferentiated induced pluripotent stem cells, The present invention also relates to a cell culture substantially consisting of hepatocytes obtained by the method.

  In recent years, transplantation therapy using cells differentiated from pluripotent stem cells has attracted attention in regenerative medicine for the purpose of regeneration and functional recovery of cells, tissues, and organs that have malfunctioned or impaired function. Pluripotent stem cells are cells that have both self-replicating ability and pluripotency to replicate themselves, and it is a major issue in regenerative medicine to induce and amplify target cells from pluripotent stem cells. is there.

  The pluripotent stem cells are known as induced pluripotent stem cells (hereinafter abbreviated as iPS cells) and embryonic stem cells (hereinafter abbreviated as ES cells). It has been. These cells are expected to be applied to regenerative medicine by transplantation therapy in which the target cells are differentiated and used.

  For example, if hepatocytes can be induced to differentiate from human iPS cells or human ES cells, development to transplantation therapy for liver failure cases is expected. The liver is mainly composed of hepatocytes that are hepatocytes, non-hepatocytes such as biliary epithelial cells, and the like. In addition to the secretion of bile, filtration and detoxification of absorbed nutrients, drug metabolism, storage of sugar and regulation of blood glucose, the liver is a vital organ that is a vital organ, such as fibrinogen, heparin, and anemia inhibitor. Therefore, liver failure in which functioning hepatocytes are extremely reduced is a fatal disease state, and in cases of liver failure, a very serious state such as bleeding tendency due to insufficient coagulation factor and hepatic coma. Hepatocyte transplantation can be a fundamental treatment for such pathologies.

  The present inventor provides a method for inducing differentiation of hepatocytes from ES cells and obtaining a cell culture substantially composed of hepatocytes from a group of cells containing the differentiated hepatocytes (Patent Documents 1 and 2). . Specifically, because hepatocytes have a series of enzymes such as glycolysis and urea cycle, we focus on the fact that they survive in a medium supplemented with the substrates galactose and ornithine, except for the glucose and arginine products. Provide a method of obtaining a cell culture consisting essentially of hepatocytes by culturing in such a medium to kill cells differentiated from embryonic stem cells into other cells other than hepatocytes doing.

  However, regarding the application of human ES cells to humans, there are immune rejection and ethical problems derived from the fact that the ES cells are embryonic stem cells derived from others.

  On the other hand, iPS cells are pluripotent stem cells derived from somatic cells such as human fibroblasts by gene recombination technology, and can be prepared from their own cells, so there are few problems such as immune rejection and regenerative medicine. Application to this is expected (Non-patent Document 1).

  As a technique for inducing differentiation of hepatocytes from human iPS cells, a method has been reported in which growth factors and / or transcription factors that promote differentiation into hepatocytes are added to the culture of human iPS cells, respectively, and introduced (non-substantially). Patent Document 2, Non-Patent Document 3, Non-Patent Document 4).

  However, iPS cells also have a problem that they may become cancerous when transplanted (Non-patent Document 5). Since undifferentiated iPS cells are mixed in a cell group containing hepatocytes differentiated from iPS cells, it is necessary to obtain differentiated hepatocytes separately from the cell group in order to apply clinically to differentiated hepatocytes. is there.

Japanese Patent No. 4759723 JP 2005-253374 A JP 2012-143229 A

Takahashi K et al., “Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors”, Cell, 2007, Vol. 131, No. 5, p.861-872. Tomizawa M et al., “Liver”, 2011, Volume 52, (Extra Number 2): A680. Inamura et al., “Efficient Generation of Hepatoblasts From Human ES Cells and iPS Cells by Transient Overexpression of Homeobox Gene HEX”, Molecular Therapy, 2011, Vol. 19, No. 2, p.400-407 . Tomizawa M et al., “Single-step protocol for the differentiation of human-induced pluripotent stem cells into hepatic progenitor-like cells”, Biomedical Reports, 2013, Volume 1, pages 18-22 (Published online on Monday, August 13, 2012 as Doi: 10.3892 / br.2012.2). Cunningham et al., “Lessons from human teratomas to guide development of safe stem cell therapies”, Nature Biotechnology, 2012, Vol. 30, pages 849-857. Tomizawa M et al., “Activin A is essential for feeder-free culture of human induced pluripotent stem cells”, Journal of Cellular Biochemistry (in press), (Published online as DOI) : 10.1002 / jcb.24395). Kakinuma S et al., “Analyses of cell surface molecules on hepatic stem / progenitor cells in mouse fetal liver”, Journal of Hepatology, 2009, Vol. 51, No. 1, p.127-138 . Sangan CB et al., “Hepatic progenitor cells”, Cell and Tissue Research, 2010, Volume 342, No. 2, p. 131-137. Yamada T et al., “In vitro differentiation of embryonic stem cells into hepatocyte-like cells identified by cellular uptake of indocyanine green”, Stem Cells, 2002, 20, 20, p.146. -154. Tomizawa M et al., “Hepatocytes deficient in CCAAT / enhancer binding protein alpha (C / EBP alpha) exhibit both hepatocyte and biliary epithelial cell character”, Biochemical and Biophysical Research Communications, 1998 Year, Volume 249, Issue 1, pages 1-5.

  An object of the present invention is to provide a method for obtaining a cell culture substantially consisting of hepatocytes from a cell group including hepatocytes induced to differentiate from iPS cells and undifferentiated iPS cells.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor has found a medium that has the effect of killing iPS cells but can maintain and proliferate hepatocytes. Then, using such a medium, a cell group containing hepatocytes differentiated from iPS cells and undifferentiated iPS cells is cultured in the medium to obtain a cell culture substantially consisting of hepatocytes. I found that I can do it. The present invention has been completed based on this finding.

  That is, the present invention is a method for obtaining a cell culture comprising hepatocytes from a group of cells containing hepatocytes induced to differentiate from iPS cells and undifferentiated iPS cells, wherein the cell groups are obtained from serum or The present invention relates to a method comprising a step of culturing in a medium having the composition shown in Table 1 or Table 2 containing a serum replacement substance.

The present invention also relates to serum or serum replacement material knockout ^trademark serum replacement; relates to the above method is a (knockout ^TM serum replacement, Invitrogen Corporation hereinafter referred to as KSR).

  The present invention further relates to the above method, wherein KSR is added to a final concentration of 10% (V / V).

  The present invention still further relates to the above method, wherein the culturing step is a step of culturing for at least 3 days.

  The present invention is also the above method, wherein the cell group containing hepatocytes differentiated from iPS cells and undifferentiated iPS cells is a cell group containing hepatic progenitor cells differentiated from iPS cells and undifferentiated iPS cells. About.

  The present invention further includes a cell group comprising hepatocytes induced to differentiate from iPS cells and undifferentiated iPS cells, wherein human iPS cells are transfected with the transcription factor FOXA2, GATA4, HEX and C / EBPα genes every 3 days. A cell containing human hepatic progenitor cells and human iPS cells obtained by inducing differentiation in a medium containing Oncostatin M, epidermal growth factor, retinoic acid, dexamethasone, insulin and transferrin as a combination of growth promoters The above method is a group.

  The present invention is also a method for obtaining a cell culture comprising hepatocytes from a cell group comprising hepatocytes induced to differentiate from iPS cells and undifferentiated iPS cells, wherein the liver is induced to differentiate from iPS cells. A cell group including cells and undifferentiated iPS cells is transfected with human iPS cells with transcription factor FOXA2, GATA4, HEX and C / EBPα every 3 days, and oncostatin M as a combination of growth promoters, Induction of differentiation in a medium containing epidermal growth factor, retinoic acid, dexamethasone, insulin and transferrin, and the final concentration of a group of human hepatic progenitor cells obtained 8 days after transfection and human iPS cells The present invention relates to a method comprising a step of culturing at least 3 days in a medium having the composition shown in Table 1 or Table 2 containing KSR added so as to be 10% (V / V).

  The present invention provides a method for obtaining a cell culture consisting essentially of hepatocytes from a cell group containing hepatocytes differentiated from iPS cells and undifferentiated iPS cells.

  The culture medium used in the method according to the present invention can kill human iPS cells that may cause carcinogenesis when transplanted, whereas hepatocytes exhibit the same viability as the optimal medium for the culture. Therefore, according to the present invention, iPS cells can be killed in a cell group including hepatocytes induced to differentiate from iPS cells and undifferentiated iPS cells, and thereby a cell culture substantially consisting of hepatocytes. Can be obtained separately. As described above, according to the present invention, in transplantation of a target cell differentiated from iPS cells, human iPS cells that may cause carcinogenesis after transplantation can be excluded. Clinical application becomes possible.

When human iPS cells (201B7, RIKEN Cell Bank) were cultured after replacing the medium with hepatocyte selection media (hereinafter abbreviated as HSM), the number of cells decreased one day after changing the medium (Day 1). It is a figure which shows having disappeared three days later (Day3) first. The HSM was a medium having the composition shown in Table 1 or Table 2 above, and a medium containing KSR was used. In the figure, each of Dialysis (+) and (-) indicates the presence or absence of KSR dialysis. Ins, Dex, Apr (+) and (−) indicate a medium containing insulin, dexamethasone and aprotinin and a medium not containing these, respectively. Magnification: 40 times, scale bar: 25 μm. (Example 1) FIG. 3 shows that when human iPS cells were cultured with the medium changed to HSM, the number of cells started to decrease 1 day after the medium was changed and all disappeared after 3 days. ○ Solid line is cultured in a medium containing KSR that is not dialyzed and insulin, dexamethasone, and aprotinin (D (-) I (+)), x solid line is KSR that is not dialyzed, and does not contain insulin, dexamethasone, and aprotinin Culture in medium (D (-) I (-)), dotted line is dialyzed KSR and culture in medium containing insulin, dexamethasone and aprotinin (D (+) I (+)), x dotted line is dialyzed The result of culture | cultivation (D (+) I (-)) in the culture medium which contains KSR but does not contain insulin, dexamethasone, and aprotinin is shown. In the figure, the horizontal axis indicates the number of days after changing the medium to HSM (Days after medium change), and the vertical axis indicates the number of cells per cell hole (Cell number / well). (Example 1) When human iPS cells are cultured with the medium changed to HSM, the colonies formed are observed to be reduced. Panels A, B, C, and D, respectively, include culture in a medium containing non-dialyzed KSR and insulin, dexamethasone, and aprotinin (D (-) I (+)), non-dialyzed KSR, insulin, dexamethasone, And culture in a medium not containing aprotinin (D (-) I (-)), culture in a medium containing dialyzed KSR and insulin, dexamethasone, and aprotinin (D (+) I (+)), and dialysis The results of culture (D (+) I (−)) in a medium containing the above-described KSR but not insulin, dexamethasone, and aprotinin are shown. Panel E shows the results of culturing with ReproFF without changing the medium to HSM, and no colony reduction was observed in such culture. Panel F is an enlargement of part of panel D, with arrows and arrowheads indicating nuclear condensation and fragmentation, respectively. Magnification: 400 times, scale bar: 25 μm (A, B, C, D, E) and 2.5 μm (F). (Example 1) It is a figure which shows that the human iPS cell raise | generated apoptosis (apoptosis), when culture | cultivating by replacing | exchanging a culture medium to HSM. Panels A, B, C, and D, respectively, include culture in a medium containing non-dialyzed KSR and insulin, dexamethasone, and aprotinin (D (-) I (+)), non-dialyzed KSR, insulin, dexamethasone, And culture in a medium not containing aprotinin (D (-) I (-)), culture in a medium containing dialyzed KSR and insulin, dexamethasone, and aprotinin (D (+) I (+)), and dialysis The results of culture (D (+) I (−)) in a medium containing the above-described KSR but not insulin, dexamethasone, and aprotinin are shown. Panel E shows the result of culturing with ReproFF without changing the medium to HSM, and no apoptosis was observed in such culture. Panel F is an enlargement of part of panel D, with arrowheads indicating nuclear condensation. Magnification: 400 times, scale bar: 25 μm (A, B, C, D, E) and 2.5 μm (F). (Example 1) It is a figure which shows that a human iPS cell was leukocyte alkaline phosphatase positive also by culture | cultivation with maintenance medium ReproFF, or after culture | cultivating by changing a culture medium to HSM. Panels A, B, C, and D, respectively, include culture in a medium containing non-dialyzed KSR and insulin, dexamethasone, and aprotinin (D (-) I (+)), non-dialyzed KSR, insulin, dexamethasone, And culture in a medium not containing aprotinin (D (-) I (-)), culture in a medium containing dialyzed KSR and insulin, dexamethasone, and aprotinin (D (+) I (+)), and dialysis The results of culture (D (+) I (−)) in a medium containing the above-described KSR but not insulin, dexamethasone, and aprotinin are shown. Panel E shows the results of culturing with ReproFF without changing the medium to HSM. Magnification: 400 times, scale bar: 25 μm. (Example 1) When human iPS cells were cultured with the medium replaced with HSM, the cells decreased after 1 day (Day 1) and changed after 3 days (Day 3). On the other hand, human primary cultured hepatocytes showed no difference between HSM culture and special hepatocyte culture medium (hereinafter abbreviated as HCM) culture. The HSM used was HSM (D (+) I (−)) containing dialyzed KSR and no insulin, dexamethasone, and aprotinin. In the figure, human iPS cells are indicated as 201B7, and human primary cultured hepatocytes are indicated as hepatocytes. Magnification: 400 times, scale bar: 25 μm. (Example 1) When human iPS cells were cultured with the medium replaced with HSM, the cells decreased after 1 day (Day 1) and changed after 3 days (Day 3). On the other hand, human primary cultured hepatocytes showed no difference between HSM culture and HCM culture. The HSM used was HSM (D (+) I (−)) containing dialyzed KSR and no insulin, dexamethasone, and aprotinin. In the figure, ◯ indicates human iPS cells cultured with HSM, and x and □ indicate human primary cultured hepatocytes cultured with HCM and HSM, respectively. The horizontal axis indicates the number of days after changing the medium to HSM (Days after medium change), and the vertical axis indicates the number of cells per plate hole (Cell number / well). (Example 1) Human iPS cells have significantly lower expression of galactokinase 1 (galaktokinase 1, GALK1), galactokinase 2 (GALK2), and ornithine transcarbamylase (OTC) compared to human fetal liver and human adult liver. (N = 3). In the figure, FF represents human iPS cells cultured with ReproFF, fetal represents human fetal liver, and adult represents human adult liver. A vertical axis | shaft shows ratio of the expression level of each enzyme with respect to the expression level of ribosomal protein L19 (RPL19) which is an internal standard. (Example 2)

  The present invention relates to a method for obtaining a cell culture consisting essentially of hepatocytes from a cell group comprising hepatocytes induced to differentiate from iPS cells and undifferentiated iPS cells.

  The method according to the present invention comprises a group of cells containing hepatocytes induced to differentiate from iPS cells and undifferentiated iPS cells, wherein L-arginine, L-tyrosine, L-cystine, D-glucose, and pyruvic acid are substantially It is characterized in that it is cultured in a medium having a composition that does not contain. L-arginine and L-tyrosine are essential amino acids and are synthesized in the liver by urea cycle from L-ornithine and phenylalanine hydroxylase complex from L-phenylalanine, respectively. L-cystine is synthesized by specifically oxidizing the sulfhydryl group of L-cysteine in the liver to form a disulfide bond. In the process of energy metabolism, D-glucose is decomposed into pyruvate, but not only D-glucose but also D-galactose and glycerol are decomposed into pyruvate by metabolic enzymes specific to the liver. Therefore, cells that do not express an enzyme related to the urea cycle or glycolysis are difficult to survive when cultured in a medium having such a composition.

  As shown in the examples described below, iPS cells consist of ornithine transcarbamylase (OTC), an enzyme related to urea cycle, and galactokinase 1, GALK1 and galacto, which are related to glycolysis. The expression level of kinase 2 (GALK2) was low, and it was killed by culturing in a medium not containing glucose and arginine to cause apoptosis. On the other hand, since fetal liver and adult liver all express OTC, GALK1, and GALK2, survival was confirmed even in culture using such a medium.

  In the present specification, “substantially free of a substance or the like” means that a substance or the like is not contained in the medium at all or does not inhibit the induction of the action of a metabolic enzyme specific to the target cell. Means included below. For example, when the concentration is below the level that can be detected by the usual analytical method, that is, when the amino acid is 1 ng / l or less, D-glucose is 10 ng / l or less, and pyruvic acid is 10 ng / l or less, it is included in this meaning. .

  Furthermore, the medium may preferably contain serum, serum replacement substances, growth factors, protease inhibitors, and / or reducing agents. Examples of growth factors include corticosteroids such as insulin and dexamethasone. An example of a protease inhibitor is aprotinin. An example of the reducing agent is mercaptoethanol.

  The serum can be exemplified by sera from cows and pigs, preferably fetal calf serum (FCS). Since serum is likely to contain the above-described components that need to be removed from the medium, it is preferable to use a serum obtained by removing low molecular weight substances by dialysis. The amount of serum added is 5 to 20 volume / volume (V / V)%, preferably 5 to 10% (V / V), more preferably 5% (V / V).

The serum substitute substance means a component that can be used in place of serum and can exert the same effect as serum on the maintenance and proliferation of cells. In view of the clinical application of the cell culture obtained by the method according to the present invention, it is desirable that the cell culture is not exposed to heterologous proteins. Therefore, in the method according to the present invention, it is more preferable to use a serum replacement substance that does not contain a heterologous protein, instead of serum. A preferable example of a serum replacement substance is Knockout ^™ serum replacement (knockout ^™ serum replacement, manufactured by Invitrogen; KSR). KSR is known to have the same effect as serum in maintaining pluripotent stem cells, and its components do not contain heterologous proteins. Other serum replacements include BD Nu-Serum ^™ replacement (Becton Dickinson), XerumFree ^™ FBS replacement (TNC BIO BV, Eindhoven, The Netherlands), CDM-HD Serum replacement (FiberCell Systmems Inc., Frederick) , MD). The amount of the serum substitute substance to be added varies depending on the serum substitute substance to be used, but can be determined by conducting simple repeated experiments as appropriate. Taking KSR as an example, the amount added is 5% to 20% (V / V), preferably 10% to 20% (V / V), more preferably 10% (V / V).

  Specific examples of the medium include those shown in the following Table 1 or Table 2 containing serum or serum replacement substances. The difference between the composition shown in Table 1 and the composition shown in Table 2 is that the former composition contains insulin, dexamethasone, and aprotinin, whereas the latter composition does not contain these substances. The presence or absence of addition of insulin, dexamethasone, and aprotinin to the medium hardly affects the effect of killing iPS cells and the effect of maintaining and proliferating hepatocytes in the medium.

In the method according to the present invention, a period of culturing a cell group containing hepatocytes induced to differentiate from iPS cells and undifferentiated iPS cells in a medium having the composition of Table 1 or Table 2 containing serum or a serum replacement substance, Preferably it is at least 3 days or longer. By culturing in such a medium for 3 days or longer, undifferentiated iPS cells contained in the cell group die. On the other hand, when cultured for more than 14 days, the morphology of hepatocytes changes. The culture period is more preferably 3 days to 14 days, further preferably about 3 days to 7 days, and even more preferably about 3 days. Culturing can be carried out at normal culturing conditions, for example, 35-40 ° C., preferably 37 ° C. under an atmosphere of 95% air and 5% CO ₂ . It is appropriate to perform the medium exchange preferably once every 1-2 days, more preferably once every two days.

  iPS cells are pluripotent stem cells derived from somatic cells such as human fibroblasts by genetic recombination techniques. A pluripotent stem cell refers to a stem cell that has long-term self-renewal ability under a predetermined culture condition and has multipotency into various cells under a predetermined differentiation-inducing condition.

  The iPS cells may be derived from any species as long as they are produced from mammalian, for example, human or mouse somatic cells, but when used for regenerative medicine such as transplantation, regenerative medicine Those produced from somatic cells derived from the target species are more preferred, and those produced from somatic cells collected from the subject itself are more preferred.

  iPS cells may be prepared by any of the commonly used methods (Non-patent Document 1, etc.). Moreover, iPS cells can be subcultured using a known method of maintaining and culturing in an undifferentiated state (Patent Document 3, Non-Patent Document 6).

  In the present specification, the term “hepatocyte” is used to mean cells including all differentiation stages in which differentiation into hepatocytes has been determined, such as hepatic progenitor cells and mature hepatocytes. Mature hepatocytes, also called mature hepatocytes, are terminally differentiated cells that express a wide variety of liver-specific functions such as cholesterol synthesis, amino acid transport activity, and G-6-P dehydrogenase activity. On the other hand, as is well known in the liver regeneration phenomenon, it has an active proliferation ability. Liver progenitor cells are reported to be cells that have the ability to proliferate actively and differentiate into hepatocytes and bile duct epithelium (Non-Patent Document 7), and small and round cells that are produced during the regeneration of the liver (Oval cell) (Non-patent Document 8), which has the ability to proliferate and differentiate into hepatocytes and bile duct epithelial cells. Hepatic progenitor cells are more proliferative than mature hepatocytes and also form bile duct epithelium, so when transplanted into the liver, they quickly form an existing liver structure and are lost more effectively than transplanting only hepatocytes. It can be expected to reproduce the liver (Non-patent Document 8). For this reason, the hepatocytes induced to differentiate from iPS cells are preferably hepatic progenitor cells induced to differentiate from iPS cells.

  Any known method can be used for inducing differentiation of hepatocytes from iPS cells. For example, methods for adding and introducing growth factors and / or transcription factors that promote differentiation into hepatocytes into human iPS cell cultures have been reported (Non-patent Document 2, Non-patent Document 3, Non-patent Document). Reference 4). Using such a method described in the previous report, hepatocytes can be induced to differentiate from iPS cells.

  For example, human iPS cells are transfected with genes for transcription factors FOXA2, GATA4, HEX and C / EBPα every 3 days, and oncostatin M, epidermal growth factor, retinoic acid, dexamethasone, insulin and transferrin as a combination of growth promoters. Human liver progenitor cells can be obtained 8 days after transfection by inducing differentiation in a medium containing (Non-patent Document 4). FOXA2, GATA4, HEX and C / EBPα are transcription factors that are expressed in fetal or adult liver cells but not in pluripotent stem cells. FOXA2, GATA4, HEX and C / EBPα are human FORKHEAD box A2 gene, human GATA4 binding protein 4 gene, HHEX, a homeobox gene expressed in human hematopoietic system, and human CCAAT enhancer binding protein alpha gene, respectively Say. The base sequences of these genes and the amino acid sequences of the proteins encoded by these genes are well known in the technical field to which the present invention belongs. Transfection can be performed using known genetic engineering techniques. The specific transfection method, the medium to be used, the culture conditions, and the like can be determined with reference to the description in the previous report (Non-patent Document 4).

  By culturing a cell group including hepatocytes induced to differentiate from iPS cells and undifferentiated iPS cells in a medium having the composition shown in Table 1 or Table 2 containing serum or a serum substitute substance, undifferentiated iPS cells are obtained. Although it is killed, hepatocytes exhibit the same viability as culture in an optimal medium for culturing hepatocytes. Therefore, when producing and transplanting hepatocytes from iPS cells, human iPS cells are killed by culturing a group of cells containing hepatocytes differentiated from iPS cells and undifferentiated iPS cells in such a medium. Thus, a cell culture substantially consisting of hepatocytes can be obtained.

  As used herein, “consisting essentially of hepatocytes” means that the ratio of hepatocytes to undifferentiated iPS cells (hepatocytes: undifferentiated iPS cells) is 100: 1 or less, preferably 1,000: 1 or less, More preferably, it means 10,000: 1 or less.

  The fact that a cell culture consisting essentially of hepatocytes was obtained by the method according to the present invention is a method utilizing the specific uptake of indocyanine green (ICG) into hepatocytes (Non-patent Document 9, etc.) Alternatively, it can be confirmed by a method for detecting expression of a gene specific to hepatocytes. For example, confirmation of differentiation induction from iPS cells to hepatic progenitor cells or hepatocytes can be confirmed by increasing production of α-fetoprotein (AFP), a marker of immature hepatocytes, in immature cells such as stem cells and progenitor cells. Upregulation of Delta like-1 (DLK-1), which regulates proliferation and differentiation of cells and suggests differentiation into hepatocytes, and / or γ-glutamyl transpeptidase (γ-), a marker of biliary epithelium Increased expression of glutamyl transpeptidase (γ-GTP) and ICG uptake reflecting drug metabolism as hepatocytes can be used as indicators (Non-patent Documents 3 and 10). However, it is not limited to these methods, and any known method can be used.

  The fact that iPS cells are not contained in the cell culture obtained by the method according to the present invention can be confirmed by measuring that the cells in the cell culture do not retain the iPS cell index. it can. The most accurate indicators of iPS cell undifferentiation include cell morphology, positive alkaline phosphatase staining, and retention of NANOG expression. That is, if alkaline phosphatase staining of cells in a cell culture is negative and / or if a decrease in NANOG expression is detected, it is determined that the cell culture does not contain iPS cells. Can do. However, it is not limited to these methods, and any known method can be used.

  The marker of differentiation-induced hepatocytes and undifferentiated iPS cells is a known genetic engineering such as polymerase chain reaction (RT-PCT) or real-time polymerase chain reaction after reverse transcription of mRNA of the target protein in the cell. Can be confirmed by an enzymatic method, or by an enzyme immunoassay (ELISA method) or an immunostaining method using an antibody against a test protein, but is not limited to these methods, and any known method should be used. Can do.

  The cell culture obtained by the method of the present invention contains substantially no cells other than hepatocytes, and it is not necessary to add a step of separating hepatocytes. Therefore, the cell culture substantially consisting of hepatocytes obtained by the method according to the present invention can be used as it is for regeneration of liver tissue. By administering the cell culture to a subject, the liver tissue of the subject is regenerated, and as a result, the function of the liver tissue is regenerated. Therefore, the cell culture obtained by the method according to the present invention is suitable for regenerative medicine including transplantation treatment for liver diseases such as fulminant hepatitis, partial hepatectomy, or liver failure that occurs during the natural course of cirrhosis. Can be used. As used herein, the term “administering” includes meanings such as injection in blood and the like, as well as meanings such as cell transplantation in which a cell culture is directly transplanted into liver tissue.

  A cell culture substantially consisting of hepatocytes obtained by the method of the present invention is used as a medicament for the treatment of liver diseases such as fulminant hepatitis, partial hepatectomy, or liver failure that occurs during the natural course of cirrhosis. Can be used. A medicament containing a cell culture obtained by the method of the present invention as an active ingredient may contain a pharmacologically acceptable physiological saline, an additive, a medium, or the like. Those without contamination are preferred.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example.

  Human iPS cells (201B7, Riken Cell Bank) and human primary cultured hepatocytes were changed from each cell's maintenance medium to hepatocyte selection medium (hereinafter abbreviated as HSM), and changes in each cell were examined. .

In this example, HSM is a medium having the composition shown in Table 1 or 2 above, and knockout ^trademark serum replacement (hereinafter abbreviated as KSR; manufactured by Life Technologies) at a final concentration of 10% (V / V). A medium containing so as to be used was used. KSR is a reagent used as a serum substitute necessary for ES cell culture, and it has been confirmed that it does not contain heterologous proteins for humans. KSR examined the effect of dialysis and non-dialysis (D (+), D (-)). In addition, the effects of the presence or absence of insulin, dexamethasone, and aprotinin (I (+), I (-)) were also examined.

(1) Human iPS cells were cultured in HSM and cell changes were examined.

Human iPS cells were cultured in Matrigel-coated culture dishes at 37 ° C. and 5% CO ₂ using ReproFF (manufactured by Reprocell) as a medium. The cultured human iPS cells were collected using Accutase (manufactured by Innovative Cell Technologies) and seeded in a Matrigel-coated 6-well plate. Matrigel coating growth factor reduced BD Matrigel ^{® (Growth factor reduced BD Matrigel TM;} Becton, Dickinson and Company , Ltd.) DMEM-F12 medium was dispensed to 0.5 ml was diluted 30-fold with (Sigma), at room temperature for 3 This was done by incubation for a period of time. At the time of seeding, Y27632 (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the medium ReproFF at a concentration of 10 M, but not added when the medium was replaced. After reaching 80% confluence, the medium was changed to HSM and further cultured.

Changes in iPS cells were observed with an optical microscope (CKX41N-31PHP, manufactured by Olympus). The results of observation with an optical microscope are shown in FIG. 1-A. In FIG. 1-A, the number of cells was counted by taking a photograph with a field of view 400 times. In the field of view of 400 times, the area per field of view is 5.62 × 10 ^-4 cm ² and the area per hole of 6-well plate is 9.4 cm ^2, so the number of cells per field of view is calculated as follows: Converted to the number of cells per cell: number of cells per hole of 6-well plate = number of cells per field × 9.4 / (5.62 × 10 ⁻⁴ ). The number of cells was measured for 5 visual fields, and the average value was calculated (FIG. 1-B).

  iPS cells started to decrease in cell number 1 day after the medium was changed to HSM, and disappeared after 3 days (FIGS. 1-A and 1-B). This result was not affected by the presence or absence of KSR dialysis or the addition of insulin, dexamethasone, and aprotinin. From this result, a medium having the composition shown in Table 1 and containing KSR, or a medium having the same composition as the composition shown in Table 1 except that it does not contain insulin, dexamethasone, and aprotinin, It became clear that human iPS cells were also killed by culturing in a medium containing.

(2) Human iPS cells were cultured in HSM, and changes in the morphology were examined by staining with hematoxylin eosin.

  Human iPS cells were seeded on Matrigel-coated 4-hole chamber slides (BD Falcon). ReproFF was used as the medium. After reaching 80% confluence, the medium was changed to HSM and further cultured. One day after the medium was changed to HSM, hematoxylin and eosin were stained according to a conventional method, and observed with an optical microscope (AX80, Olympus). After 2 days of medium change, the number of cells was extremely reduced and analysis became difficult, so 1 day after the medium change was selected as the analysis day.

  By changing the medium to HSM and culturing, reduction of colonies formed in iPS cell culture was observed (panel A, panel B, panel C, and panel D in FIG. 2). This result was not affected by the presence or absence of KSR dialysis or the addition of insulin, dexamethasone, and aprotinin. Moreover, nuclear condensation (Panel F in Fig. 2, arrow) and fragmentation (Panel F in Fig. 2, arrowhead) were observed under all conditions. On the other hand, no colony reduction was observed in the group cultured with ReproFF without changing the medium (panel E in FIG. 2).

  From the above results, it was suggested that iPS cells may be in apoptosis in the group whose medium was changed to HSM.

(3) Human iPS cells were cultured in HSM, and whether or not the cells had undergone apoptosis was analyzed.

  Human iPS cells were seeded on Matrigel-coated 4-hole chamber slides (BD Falcon). ReproFF was used as the medium. After reaching 80% confluence, the medium was changed to HSM and further cultured. One day after the medium was changed to HSM, the cells were stained using an apoptosis in situ detection kit (Apoptosis in situ detection kit, manufactured by Wako Pure Chemical Industries) according to the procedure manual, and analyzed for the presence or absence of apoptosis of the cells. This kit is based on the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method.

  TUNEL positive cells were observed in the group cultured with the medium changed to HSM (Panel A, Panel B, Panel C, Panel D, and Panel F (arrowheads) in FIG. 3). On the other hand, TUNEL positive cells were not observed in the group cultured with ReproFF without changing the medium (panel E in FIG. 3).

  From the above results, it was suggested that iPS cells undergo apoptosis when cultured with HSM.

(4) It was analyzed whether human iPS cells cultured in HSM were in an undifferentiated state.

  Human iPS cells were seeded in a 6-well plate coated with Matrigel as described above. After reaching 80% confluency, the medium was changed to HSM, and after 1 day, staining was performed using a leukocyte alkaline phosphatase kit (Leukocyte Alkaline Phosphatase Kit, Sigma-Aldrich) according to the procedure manual, and light microscope (CKX41N-31PHP, Olympus) Observed).

  Human iPS cells were positive for leukocyte alkaline phosphatase after culturing under any conditions (FIG. 4). This suggests that human iPS cells remain undifferentiated when cultured in HSM.

(5) Human iPS cells and human primary cultured hepatocytes (Clontech) were cultured in HSM, and changes in the cells were compared. HSM has the same composition (the composition in Table 2 above) except that it does not contain insulin, dexamethasone and aprotinin, and the dialyzed KSR has a final concentration of 10% (V / V). HSM (HSM D (+) I (-)) was used.

Human primary cultured hepatocytes were cultured according to Clontech's protocol. In other words, ² × 10 ⁵ hepatocytes were seeded per 1 cm ² in a 24-well plate (CellBIND, manufactured by Corning) coated with Matrigel by the thin gel method. Matrigel coating by the thin gel method was performed by dispensing 50 μl of growth factor reduced BD matrigel phenol red free (Becton Dickinson) per cm ^{2 and} incubating at 37 ° C. for 30 minutes. The maintenance culture was performed using a hepatocyte culture medium (hereinafter abbreviated as HCM) manufactured by Clontech.

  Human iPS cells were seeded in 24-well plates and maintained in the same manner as in (1) above.

Both human primary cultured hepatocytes and human iPS cells were observed with an optical microscope (CKX41N-31PHP, Olympus) after changing the medium to HSM after 1 day of culture. Specifically, the number of cells was counted by taking a photograph with a field of view of 400 times. In the field of view of 400 times, the area per field of view is 5.62 × 10 ^-4 cm ² and the area per hole of 24 holes plate is 2 cm ^2, so the number of cells per field of view is calculated as follows: Converted to the number of cells per cell: Number of cells per well of 24-well plate = number of cells per field × 2 (5.62 × 10 ⁻⁴ ). The number of cells was measured for 5 visual fields, and the average value was calculated.

  Human iPS cells decreased in number when cultured with HSM, while human primary cultured hepatocytes showed no difference between the group cultured with HSM and the group cultured with HCM (FIGS. 5-A and 5-). B).

  Expression of galactokinase 1 (galaktokinase 1, GALK1), galactokinase 2 (GALK2), ornithine transcarbamylase (OTC) was compared between human iPS cells and human fetal liver and human adult liver.

  RNA was extracted from human iPS cells using Isogen (Nippon Gene, Tokyo, Japan). First-strand cDNA is synthesized from 5 g of total RNA (Total RNA) using Superscript III (SuperScript III; Life Technologies, Grand Island, NY) using oligo dT (Oligo dT) as a primer. did. Human fetal liver and human adult liver RNA were purchased from Clontech.

Real-time quantitative PCR (Real-time quantitative PCR) is, Fast SYBR ^{registered trademark} Green Master Mix ^{(Fast SYBR (R) Green Master} Mix: Life Technologies , Inc.) Miniopuchikon ^trademark real-time PCR Detection system using a (MiniOpticon ^TM real-time PCR detection system: Bio-Rad, Hercules, CA) and analyzed. Real-time quantitative PCR was performed in 40 cycles, with denaturation being 5 seconds and annealing-extension being 1 cycle in 5 seconds. Ribosomal protein L19 (RPL19) was measured as an internal control.

The base sequence of the primer set used for PCR and the expected size of the PCR product obtained using the primer set are shown below.
(A) RPL19 amplification primer:
5'-CGAATGCCAGAGAAGGTCAC (SEQ ID NO: 1) and 5'-CCATGAGAATCCGCTTGTTT (SEQ ID NO: 2) ((GeneBank Accession Number: BC000530, expected size of PCR product: 157 bp)
(B) GALK1 amplification primer:
5'-TGCTGTGCCTGGGGTTTATG (SEQ ID NO: 3) and 5'-GCTGCTTGAGAGAGGTAGAAGGTG (SEQ ID NO: 4) (GeneBank Accession Number: NM_000154, expected size of PCR product: 153 bp)
(C) GALK2 amplification primer:
5'-TCACGACTTACTGGAGCAGGATG (SEQ ID NO: 5) and 5'-CAAAACCAAAGCCCCACCTC (SEQ ID NO: 6) (GeneBank Accession Number: NM_002044, expected size of PCR product: 177 bp)
(D) OTC amplification primers:
5'-GGACATTTTTACACTGCTTGCCC (SEQ ID NO: 7) and 5'-TCCACTTTCTGTTTTCTGCCTCTG (SEQ ID NO: 8) (GeneBank Accession Number: BC107153, expected size of PCR product: 105 bp)

  The expression levels of OTC, GALK1, and GALK2 were significantly lower in human iPS cells than in human fetal liver and human adult liver (FIG. 6).

  From the above results, it can be considered that iPS cells were killed by culturing in HSM not containing glucose and arginine because the expression levels of OTC, GALK1, and GALK2 were low. In other words, when cultured in HSM, iPS cells remain undifferentiated and are thought to cause apoptosis and die. On the other hand, since fetal liver and adult liver all express OTC, GALK1, and GALK2, it can be considered that they could survive even in culture with HSM.

  According to the present invention, a cell culture substantially consisting of hepatocytes can be obtained from a cell group comprising hepatocytes induced to differentiate from iPS cells and undifferentiated iPS cells. The cell culture substantially consisting of hepatocytes obtained by the method of the present invention excludes human iPS cells that may become carcinogenic after transplantation, and is used for regeneration of liver tissue by administration to an individual as it is. Can be used in regenerative medicine.

SEQ ID NO: 1: An oligonucleotide designed as a primer for amplifying the ribosomal protein L19 gene.
SEQ ID NO: 2: Oligonucleotide designed as a primer for amplification of ribosomal protein L19 gene.
SEQ ID NO: 3: Oligonucleotide designed as a primer for amplification of galactokinase 1 gene.
SEQ ID NO: 4: Oligonucleotide designed as a primer for amplification of galactokinase 1 gene.
SEQ ID NO: 5: oligonucleotide designed as a primer for amplification of galactokinase 2 gene
SEQ ID NO: 6: oligonucleotide designed as a primer for amplification of galactokinase 2 gene
SEQ ID NO: 7: An oligonucleotide designed as a primer for amplifying the ornithine transcarbamylase gene.
SEQ ID NO: 8: oligonucleotide designed as a primer for amplification of ornithine transcarbamylase gene

Claims (7)

A method for obtaining a cell culture comprising hepatocytes from a group of cells including hepatocytes induced from induced pluripotent stem cells (hereinafter abbreviated as iPS cells) and undifferentiated iPS cells. A method comprising culturing the cell group in a medium having the composition shown in Table 1 or 2 below containing serum or a serum replacement substance.
Serum or serum replacement material knockout ^trademark Serum Replacement (knockout ^TM serum replacement; hereinafter abbreviated as KSR) The method according to claim 1 is.   3. The method according to claim 2, wherein the KSR is KSR added to a final concentration of 10% (V / V).   4. The method according to any one of claims 1 to 3, wherein the culturing step is a step of culturing for at least 3 days.   5. The cell group comprising hepatocytes differentiated from iPS cells and undifferentiated iPS cells is a cell group comprising hepatic progenitor cells differentiated from iPS cells and undifferentiated iPS cells. Or the method according to claim 1.   A group of cells containing hepatocytes induced to differentiate from iPS cells and undifferentiated iPS cells, human iPS cells are transfected with the transcription factor FOXA2, GATA4, HEX and C / EBPα genes every 3 days, thereby promoting growth. A cell group comprising human hepatic progenitor cells and human iPS cells obtained by inducing differentiation in a medium containing Oncostatin M, epidermal growth factor, retinoic acid, dexamethasone, insulin and transferrin as a combination of 5. The method according to any one of 1 to 4. A method for obtaining a cell culture comprising hepatocytes from a group of cells including hepatocytes induced from induced pluripotent stem cells (hereinafter abbreviated as iPS cells) and undifferentiated iPS cells. A group of cells including hepatocytes differentiated from iPS cells and undifferentiated iPS cells transfect human iPS cells with the genes for transcription factors FOXA2, GATA4, HEX and C / EBPα every 3 days. Induction of differentiation in a medium containing Oncostatin M, epidermal growth factor, retinoic acid, dexamethasone, insulin and transferrin as a combination of growth promoters, human hepatic progenitor cells obtained 8 days after transfection, and human the cell group containing the iPS cells, the following Table 1 with added knocked ^trademark serum replacement to a final concentration of 10% (V / V) or in a medium with the composition shown in Table 2 Which comprises the step of culturing 3 days even without.